EFFICIENT METHOD FOR CONSTRUCTING COMPREHENSIVE MURINE FAB ANTIBODY LIBRARIES DISPLAYED ON PHAGE

We have developed efficient methodologies for construction and express ion of comprehensive phage display libraries of murine Fab antibody fr agments in E.coli cells. Our methods optimize several critical steps o f the polymerase chain reaction (PCR) amplification of transcripts of the re-arranged immunoglobulin genes and of their subsequent assembly and expression: Firstly, we have designed exhaustive sets of PCR prime rs of low degeneracy for the amplification of transcripts of the Fab r egion of the heavy and light-chain genes. These primers proved effecti ve in amplification of Fab gene fragments from a large panel of hybrid oma cell lines of different specificity and family sub-type. Secondly, we have developed a 'jumping PCR' technique that effectively assemble d and recombined the amplified heavy and light-chain gene fragments in to a bi-cistronic operon. Thirdly, we have constructed expression vect ors for insertion of the combinatorial Fab gene-cassette in fusion wit h a truncated version of the phage surface protein, gIIIp. The heavy c hain and the light chain-gIII fusion are transcribed as a polycistroni c mRNA from the lacZ promoter and efficient transcriptional control is provided by wildtype lacI present on the vector. The utility of the s ystem was demonstrated by isolating several antigen-binding clones fro m hybridomas and libraries made from immunized mice.